Cell Biology of Bioactive Peptide Secretion

生物活性肽分泌的细胞生物学

基本信息

批准号：
7583470
负责人：
RICHARD E MAINS
金额：
$ 49.62万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-09-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7583470
关键词：
ARHGEF1 gene Actins Address Affect Alzheimer&apos s Disease Anabolism Binding Biogenesis Biological Assay CRH gene Cell Culture Techniques Cell Nucleus Cell physiology Cells Cellular biology Complex Coronary Arteriosclerosis Corticotropin Cytoplasmic Granules Cytoskeleton DH Domain Data Endocrine Enzymes Epigenetic Process Essential Genes Exons Face Family Fluorescence G Protein-Coupled Receptor Signaling Gene Expression Genes Genetically Engineered Mouse Growth Guanine Nucleotide Exchange Factors Guanosine Triphosphate Health Hippocampus (Brain)Hormones Human In Vitro Individual Injection of therapeutic agent Integral Membrane Protein Knock-out Knockout Mice Knowledge Link Lipids Mediating Membrane Membrane Lipids Membrane Proteins Mixed Function Oxygenases Mus Mutation Neurophysiology - biologic function Nuclear Partner in relationship Pathway interactions Patients Peptides Phosphotransferases Physiological Pituitary Gland Pituitary Hormones Play Pro-Opiomelanocortin Production Protein Isoforms Proteins Proteomics RNA Splicing Recycling Regulation Role SH3 Domains Schizophrenia Secretory Vesicles Signal Transduction Site Somatotropin Spectrin Structure Subfamily lentivirinae Swelling Testing Variant Vasopressins Vesicle cell type early onset gel electrophoresis genetic analysis genetic linkage analysis improved in vitro testing in vivo inhibitor/antagonist mutant neoplastic cell neurotransmission peptide hormone peptidylglycine alpha-amidating monooxygenase promoter protein complex protein protein interaction public health relevance recombinase restraint stress rho rho GTP-Binding Proteins secretion process tool trafficking tumor two-dimensional vacuolar H+-ATPase

项目摘要

DESCRIPTION (provided by applicant): Proper control of the synthesis, storage and secretion of bioactive peptides is crucial to normal endocrine and neural function. Over the past several decades, we delineated the cell-type specific enzymatic steps leading from proopiomelanocortin (POMC) to its many product peptides. We selected one of these enzymes, peptidylglycine 1-amidating monooxygenase (PAM), as a focus of our studies because it is an integral membrane protein that can communicate information about the lumen of the regulated secretory pathway to cytosolic machinery and to the nucleus. Our focus on PAM led to the discovery of Kalirin, a GDP/GTP exchange factor (GEF) for small GTP binding proteins of the Rho family, that interacts with the cytosolic domain of PAM. Linkage studies associating Kalirin with coronary artery disease, the decrease in Kalirin expression associated with elevated iNOS in Alzheimer disease hippocampus, and the identification of Kalirin as one of the proteins essential for Ras-mediated epigenetic silencing of gene expression, motivate our studies of this complex, multidomain protein. By flanking an exon common to the major splice variants of Kalirin with loxp sites, Kalirin conditional knockout mice (KalCKO/CKO) and mice lacking the major isoforms of Kalirin (KalKO/KO) were created. While not yet well characterized, it is clear that normal storage and secretion of pituitary hormones require pituitary Kalirin. Using lentiviruses or mating with mice in which expression of Cre recombinase is driven by the POMC or growth hormone promoter, Kalirin expression in corticotropes or somatotropes will be eliminated. POMC and GH synthesis, processing and secretion will be evaluated in vivo and in cell culture. The hypothesis that G1q-mediated activation of the second GEF domain of Kalirin plays a key role in the ability of corticotropes to respond to specific secretagogues will be tested in vitro and in vivo. Endocytic trafficking of PAM will be evaluated to determine the role of Kalirin in recycling granule membrane proteins and PAM-mediated nuclear signaling. Proteomic analysis will be carried out on isolated immature granules, whose formation is regulated by Kalirin, and on isolated pituitary granules formed in the absence of Kalirin. Assays to detect activation of Rho GEFs and their effectors will be used to understand their physiological role in pituitary hormone secretion. Finally, we will use knowledge of its individual domains to test the hypothesis that Kalirin functions as a modular machine, coordinating multiple aspects of granule biogenesis and release. In particular, the physiological importance of the alternate N-termini of Kalirin, the ability of Kalirin to inhibit iNOS, and the ability of Kalirin to integrate signals from multiple pathways will be assessed in vivo and explored mechanistically in cell culture. PUBLIC HEALTH RELEVANCE: Precise control of the biosynthesis, storage and secretion of bioactive peptides requires the coordinate control of many different cellular processes. Genetically engineered mice were used to determine that Kalirin, a large protein with domains that allow it to interact with immature secretory granules, regulate the actin cytoskeleton, bind to lipid membranes and respond to multiple protein/protein interactions, plays an essential role in peptide hormone release. Mutations in the Kalirin gene or changes in Kalirin expression have been correlated with early-onset coronary artery disease, schizophrenia and Alzheimer Disease, making a better understanding of the functions of this complex protein relevant to human health.

描述（申请人提供）：生物活性肽的合成、储存和分泌的正确控制对于正常的内分泌和神经功能至关重要。在过去的几十年里，我们描述了从阿黑皮素原 (POMC) 到其许多产物肽的细胞类型特异性酶促步骤。我们选择其中一种酶，肽基甘氨酸 1-酰胺化单加氧酶 (PAM) 作为我们研究的重点，因为它是一种完整的膜蛋白，可以将有关受调节的分泌途径的内腔信息传达到胞质机器和细胞核。我们对 PAM 的关注导致了 Kalirin 的发现，它是 Rho 家族小 GTP 结合蛋白的 GDP/GTP 交换因子 (GEF)，可与 PAM 的胞质结构域相互作用。 Kalirin 与冠状动脉疾病的关联研究、阿尔茨海默病海马中与 iNOS 升高相关的 Kalirin 表达减少以及 Kalirin 被鉴定为 Ras 介导的表观遗传基因表达沉默所必需的蛋白质之一，这些都激发了我们对这一复合物的研究，多结构域蛋白。通过在 Kalirin 主要剪接变体共有的外显子侧翼添加 loxp 位点，创建了 Kalirin 条件性敲除小鼠 (KalCKO/CKO) 和缺乏 Kalirin 主要亚型的小鼠 (KalKO/KO)。虽然尚未得到很好的表征，但很明显，垂体激素的正常储存和分泌需要垂体 Kalirin。使用慢病毒或与由 POMC 或生长激素启动子驱动 Cre 重组酶表达的小鼠交配，促肾上腺皮质激素或生长激素中的 Kalirin 表达将被消除。 POMC 和 GH 的合成、加工和分泌将在体内和细胞培养中进行评估。 G1q 介导的 Kalirin 第二个 GEF 结构域的激活在促肾上腺皮质激素对特定促分泌素作出反应的能力中发挥关键作用的假设将在体外和体内进行测试。将评估 PAM 的内吞运输，以确定 Kalirin 在回收颗粒膜蛋白和 PAM 介导的核信号传导中的作用。将对分离的未成熟颗粒（其形成受 Kalirin 调节）和在没有 Kalirin 的情况下形成的分离的垂体颗粒进行蛋白质组分析。检测 Rho GEF 及其效应器激活的检测将用于了解它们在垂体激素分泌中的生理作用。最后，我们将利用其各个领域的知识来检验 Kalirin 作为模块化机器的假设，协调颗粒生物发生和释放的多个方面。特别是，Kalirin 的替代 N 末端的生理重要性、Kalirin 抑制 iNOS 的能力以及 Kalirin 整合来自多个途径的信号的能力将在体内评估并在细胞培养中进行机制探索。公共卫生相关性：精确控制生物活性肽的生物合成、储存和分泌需要协调控制许多不同的细胞过程。基因工程小鼠被用来确定 Kalirin（一种大型蛋白质，其结构域使其能够与未成熟的分泌颗粒相互作用、调节肌动蛋白细胞骨架、与脂质膜结合并对多种蛋白质/蛋白质相互作用做出反应）在肽激素中发挥重要作用发布。 Kalirin 基因的突变或 Kalirin 表达的变化与早发性冠状动脉疾病、精神分裂症和阿尔茨海默病相关，从而更好地了解这种与人类健康相关的复杂蛋白质的功能。